Various methods to obtain quantitative information of the degree of asymmetry of native and reconstituted respiring membranes were explored. In oxygen pulse-induced pH jumps, the measured dH ion/d0 ratio for spheroplasts was three with D-lactate and four with succinate as energy sources. With reconstituted succinoxidase vesicles in the presence of PCCP, dH ion/d0 was about 0.7. The FCCP seemed to be required for H ion transport. The development of a membrane potential in the reconstituted vesicles was indicated by the necessity for having valinomycin and K ion present to allow proton ejection and by flow dialysis using valinomycin and 86Rb.